The long-term goal of the proposed research is to understand how a network of cyclindependent kinases (CDKs) and upstream CDK-activating kinases (CAKs) coordinates cell division with gene expression and the maintenance of genome integrity in a model eukaryotic organism, the fission yeast Schizosaccharomyces pombe. In fission yeast, as in multicellular organisms, one kinase activates the major cell-cycle CDK (Cdk1) to drive cell division and phosphorylates RNA polymerase II to regulate the transcription cycle. That enzyme-Mcs6, which is homologous to human Cdk7-works together with another CDK, Cdk9, to control gene expression programs linked to the cell cycle, and to coordinate synthesis of RNA molecules with their processing to produce mature messenger RNAs (mRNAs) that can be translated into proteins. S. pombe also contains a second CAK, Csk1, which is dispensable for viability, but which activates Cdk1, Mcs6 and Cdk9. Cells lacking Csk1 have growth defects and are hypersensitive to DNA-damaging agents and replication inhibitors. The CAK-CDK network therefore signals through multiple pathways, and plays critical regulatory roles in gene expression and genomic surveillance, in addition to its canonical function in promoting cell proliferation. We will combine biochemical and genetic approaches to dissect the functions and targets of the CAK-CDK network in fission yeast. By introducing a genetically engineered version of each CDK that is susceptible to inhibition by a specially designed small molecule, we are able to switch kinase activity off in vivo, and measure the consequences for cell proliferation and gene expression. The same manipulation also allows us to identify the protein substrates of the targeted kinase. The specific aims are: [unreadable] 1. To investigate coordination between Mcs6 (Cdk7) and Cdk9, which act sequentially and in [unreadable] concert to control expression of select genes. We will investigate whether Cdk9 recruitment or [unreadable] activity in transcription elongation complexes depends on prior function of Mcs6. [unreadable] 2. To investigate the coupling of transcription (synthesis) of mRNAs with their maturation by a [unreadable] complex containing Cdk9 and an essential mRNA-processing enzyme, the mRNA 5'-cap [unreadable] methyltransferase Pcm1. [unreadable] 3. To identify new targets and functions of CAKs and CDKs in the DNA damage response. [unreadable] PUBLIC HEALTH RELEVANCE: The integrating function of the CAK-CDK network is conserved in human cells, and potentially disrupted in cancer, where impaired control of cell proliferation, derangement of gene expression and damage to the genome all contribute to the initiation and progression of tumors. If we are to block or inhibit functions of this pathway in order to stop growth and division of tumor cells, an understanding of its multiple functions will be needed to avoid toxicity to normal tissue. The powerful genetic tools available in fission yeast, and the fundamental conservation of pathway function and organization with humans, make S. pombe the ideal model system in which to perform this work. [unreadable] [unreadable] [unreadable]